Correlation of the Production of Plasminogen Activator with Tumor Metastasis in Bi 6 Mouse Melanoma Cell Lines

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[CANCER RESEARCH 40, 288-292, February 1980 0008-5472/80/0040-0000$02.00 Correlation of the Production of Plasminogen Activator with Tumor Metastasis in Bi 6 Mouse Melanoma Cell Lines

Bosco Shang Wang,2 Gerard A. McLoughlin,3 Jerome P. Richie, and John A. Mannick

Deportment of Surgery, Peter Bent Brigham Hospital (B. S. W., G. A. M., J. P. R., J. A. M.j, and Department of Pathology (B. S. W.j, Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT circulating cancer cells were easily detected in patients whose blood had a higher level of FA. The correlation between the production of plasminogen ac Although the FA of a tumor has been suggested as correlat tivator (PA) of tumors and their metastatic potential was stud ing with its metastatic potential, direct and quantitative evi ied. Bi 6 melanoma cells and â€˜â€˜Bi6 mets' â€˜cells (harvested from dence to support this hypothesis is lacking. Therefore, we have the pulmonary metastatic nodules of C57BL/6J mice bearing investigated this issue by comparing the FA of the Bi 6 mouse Bi 6 isografts) were examined with respect to their fibrinolytic melanoma and several of its sublines varying in their potential activity (FA) in tissue culture. Bi 6 mets cells had a significantly for forming metastases. higher FA than did Bi 6 cells. Fl (a Bi 6 subline with a lower incidence of metastasis) and Fl 0 (a highly metastatic Bi 6 subline) were also studied. Fl 0 cells produced more FA than MATERIALS AND METHODS did Fi cells. The difference between the FA's of these tumors Tumors. Bi 6 melanoma(The JacksonLaboratory,Bar Har was due to differences in their PA production. Significant bor, Maine) was maintained by serial transplantation either in differences in PA production between Fi and Fl 0 could be adult male C57BL/6J mice or in tissue cultures. â€˜â€˜Bi6metsâ€• consistently observed when i O@or more cells were cultured cell line was established in tissue culture by explantation of for at least 24 hr. The cell-free supernatants harvested from from 10 to 15 metastatic melanoma nodules from the lungs of 72-hr cultures of Fi 0 cells had a higher FA than those har C57BL/6J mice that had been inoculated s.c. with 1O@Bi6 vested from Fl cultures. Results suggest that a quantitative cells into the hind limbs 4 weeks previously. BI 6 mets subline difference in PA production between these 2 melanoma sub was further characterized as a melanoma by the presence of lines does exist and that it may contribute to their different melanin in the cytoplasm. The cell lines Fl and Fl 0 (kindly metastatic potential. supplied by Dr. I. J. Fidler, Frederick, Md.) were variants of the Bi 6 melanoma originally selected by explantation of succes INTRODUCTION sive generations of pulmonary metastatic cells, thus yielding 2 sublines with widely different metastatic potentials (6). Both Fl Most malignant tumors share a general pathogenesis of and Fl 0 cells have been maintained by weekly passages in blood-borne metastases. This process consists of local invasive tissue cultures with Roswell Park Memorial Institute Tissue growth of the primary tumor followed by vasculanization of the Culture Medium 1640 (Grand Island Biological Company, tumor mass and subsequent spread of tumor cells into the Grand Island, N. V.) supplemented with 10% heat-inactivated host's circulatory systems. Tumor cells that survive in the fetal calf serum, 2 mM L-glutamine (Grand Island Biological bloodstream may lodge at a distant site and develop into a Company), 100 units of penicillin per ml, and 100 @xgofstrep metastatic colony. Whether or not such a metastasis forms tomycin per ml over a period of 2 yr in our laboratory. depends upon a number of factors, among which the FA4 of Preparation of â€˜2@I-FibrIn-coatedTubes.A modified tech the tumor cells is considered to be important. nique of Moroz and Gilmore (16) was used. Two mg of 1251.. It seems logical, although still controversial, that tumors fibrinogen (specific activity, @l40Ci/mI; Abbott Laboratories, possessing a high FA should be able to digest surrounding North Chicago, III.) and 300 @gofpurified human plasminogen, fibrin and penetrate into the circulation much more easily than prepared by L-lysine Sepharose affinity chromatography (4), do tumors containing less FA. This theory is supported by a were dissolved in 100 ml of 0.01 M phosphate buffer (pH 7.4). study in which fibninolysis induced by prolonged treatment with To each 5-mI polystyrene tube, 0.2 ml of this mixture was heparin markedly increased metastatic spread in mice without added. Tubes were incubated at 37Â°with constant rotation for altering the primary tumor growth (22). A high level of FA has 3 hr. Tubes were washed 3 times with 0.05 M Tnis-O.1 M NaCI been observed in several malignant tumor cell lines (i 1â€”i3, (pH 7.4) buffer, and 6.25 mg of human serum albumin (Mas 20, 24) and also in normal embryo fibroblast cells, but only sachusetts Public Health Biological Laboratories, Boston, after these cells were transformed by oncogenic viruses (21, Mass.) in 0.5 ml of bufferwere added and incubatedat 25Â°for 23, 25). Clinically, Newstead et a!. (i 7). studied a group of 30 mm to occupy all unbound polystyrene sites. Tubes were patients with carcinoma of the colorectum and found that washed twice, and 5 units of thrombin (Topastasin; Roche Laboratories, Nutley, N. J.) in 0.1 ml were added and incubated I Supported by USPHS Grant CA21 644. at 37Â°for an additional 5 mm. Tubes were again washed 3 2 To whom requests for reprints should be addressed at the Peter Bent Brigham Hospital, 721 Huntington Avenue, Boston, Mass. 02115. times and were stored by filling with 4.5 ml of Tris-NaCI buffer 3 Recipient of Wellcome Travel Trust Grant. containing 0.1 % sodium azide at 4Â°.Thesetubes were washed 4 The abbreviations used are: FA, fibrinolytic activity; PA, plasminogen acti vator. 3 times with Dulbecco's phosphate-buffered saline (Grand Received March 16, 1979; accepted October 25, 1979. Island Biological Company) prior to use. The average cpm of

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1980 American Association for Cancer Research. PA Production and Tumor Metastasis newly prepared tubes were from 50,000 to 80,000, varying higher FA in vitro than did Bi 6 cells, even though both tumors from batch to batch. were harvested from the same animals and BI 6 mets were Fibrinolytic Activity of Tumor Cells In Vitro. Melanoma cells originally derived from Bl 6. were prepared either from C57BL/6J mice by the method of Comparison of Metastatic Potential and FA of Fl and FlO Hammond eta!. (10) or from tissue culture by trypsin treatment. Melanoma Sublines. In order to determinewhether or not the Cells were washed 3 times to remove trypsin completely and FA of Bi 6 sublines had a connection with their metastatic were then suspendedin complete Roswell Park Memorial In characteristics, 2 well-established melanoma sublines, Fl and stitute Tissue Culture Medium 1640 containing 10% fetal calf Fl 0, were used for subsequent studies. A group of mice were serum (from which plasmmnogenwas completely depleted by given injections of either Fl or Fl 0 cells into the left hind limbs. preheating to 56Â°for at least 45 mm twice), 2 mM L-glutamine, Ten days later, limbs were amputated after tumors became 100 units of penicillin per ml, and 100 @.sgofstreptomycin per palpable. Animals were killed at 6 weeks, and their lungs were ml. Cells were counted with a hemocytometer, and the viability removed and examined for evidence of metastases. As mdi was always greater than 98%, as was determined by trypan cated in Table 2, Fl had a low tendency of spontaneous blue dye exclusion. One ml of cell suspension with an appro pulmonary metastases as compared to Fl 0, which was highly priate number of tumor cells was added to each 125I-fibrin metastatic. This is in agreement with a previous observation by coated tube and was incubated at 37Â°in a 10% CO2 water Fidler (6). Furthermore, both tumor cells were obtained from saturated atmosphere for 24 hr. Tubes were centrifuged at culture flasks, and 1O@cellsin 1 ml of medium were immediately 800 x g for I 0 mm, and 0.2 ml of culture supernatant was added to each 125l-fibrin-coated tube. After incubation for 24 removed by a micropipet (Oxford Labs, Inc., Foster City, Calif.) hr, tubes were centrifuged, and 0.2 ml of supernatant was and counted in a gamma counter (Packard Instrument Co., removed and counted in a gamma counter. As shown in Table Downers Grove, Ill.). In some experiments, 10 x 1O@tumor 3, Fl 0 tumor cells had a significantly higher FA than did Fl cells were cultured in 20 ml of complete medium in a Falcon tumor cells in all 5 experiments performed (Experiments 1 to No. 3024 culture flask and were incubated for 72 hr. Cells 5). Experiments 6 to 9 were performed by incubating cell-free were removed by centrifugation, and the supernatant was supernatants harvested from 72-hr cell cultures of Fl or Fl 0. filtered through a 0.45-sm Millipore filter (Millipore Corp., Bed The FA of supernatants produced by Fl 0 was again signifi ford, Mass.). One ml of this cell-free supernatant was added to cantly greater than that of supernatants produced by Fl in 3 of each 125l-flbrmn-coatedtube,tubes were incubated for an ad 4 experiments. No significant degradation of â€˜251-fibrinwas ditional 24 hr, and 0.2 ml of this supernatant was again re observed when the plasminogen was previously removed by moved and counted in a gamma counter. The means of cpm heating the tubes to 56Â°for 30 mm, which destroyed plasmin were calculated from triplicate culture tubes, and the signifi ogen (14) but had no effect on the 125l-fibrin, since almost cance of the difference between means was determined by 100% of the radioactive fibrin could be recovered from these Student's t test. Table 2 RESULTS melanomasNo. Pulmonary metastases of Fl and FlO metastasesFlFl Comparison of FA between B16 and B16 mets. A group of of tumor05 cells Inoculated8Pulmonary C57BL/6J mice were given injections of 1O@Bl6 melanoma xiO@0110b7/91 cells into their hind limbs. Four weeks later, the mice were x i0@1/98/9 killed and Bi 6 tumor cells were obtained from the primary a C57BL/6J mice were Inoculated in their left hind limbs with either Fl or tumor isografts. Pulmonary metastatic nodules from the same Fl 0 cells. Ten days later the limbs were amputated. All mice were killed at 6 mice were explanted into tissue culture and were designated weeks, and their lungs were examined for evidence of metastases. as the â€œBi6metsâ€•subline.Original explants of both Bl 6 and b Number of animals with pulmonary metastases per total number of animals. BI 6 mets cells were incubated in â€˜25l-fibrin-coatedtubesfor 72 hr, and degraded 125l-fibrinwhichwas released into the Table 3 culture medium was measured in a gamma counter. As shown Production of FA by Fl and FlO melanomas in Table 1, 1O@Bl6 mets cells released a significantly larger â€˜25i-flbrinpFlFlO11782.6of released amount of â€˜251-fibrinfromtubes than did the same number of Experiment8cpm BI 6 cells (2966.6 versus 771 .3; p < 0.01 ). A greater FA of 343.7<0.0121754.3Â±3333b3472.6 Â± Bl 6 mets cells than that of Bl 6 cells was similarly seen when 483.0<0.0131300.3Â±227.12956.6 Â± 1O@cellswere tested (4653.0 versus 1723.0; p < 0.01 ). These 965.00.01421 Â±151.73660.0 Â± 297.8<0.0151732.000.3 Â±278.23531 .6 Â± data clearly indicated that Bl 6 mets cells had a significantly 400.0<0.0161401.0Â±178.63291.6 Â± 520.8<0.0173248.0Â± 85.23618.3 Â± 816.1<0.0582440.6Â±482.85797.6 Â± 1012.70.0593063.0Â±472.24207.3 Â± Table 1 380.6NSC10278.3 Â±946.14590.6 Â± melanomasNo.Production of FA by 81 6 and 81 6 mets Â± 31.6223.3 Â± 23.6NS

â€˜25l-fibrinpB16of released a Long-term cultured Fl and Fl 0 were used for examining forFA. Experiments of tumor 1 to 5 were performed by culturing 1O@tumorcells in â€˜25i-fibrin-coatedtubesfor cellscpmBl6Metsios771.3 24 hr. Experiments 6 to 9 were performed by incubating cell-free supernatants 639.8<0.011061723.0Â±1280a 2966.6 Â± harvested from tumor cell cultures. Experiment 10 was performed by adding cell free supernatants to tubes from which the plasminogen was removed by pre 1262.4<0.01M@ Â± 73.5 4653.0 Â± heating the tubes at 56Â°for 30 mm. b Means Â±S.D. of triplicate culture tubes. Â±S.D.of triplicate culture tubes. C NS, not significant.

FEBRUARY1980 289

treated tubes by trypsin digestion (data not shown). These Time Course of PA Production by Fl and FlO Cells. Cells results suggested that the FA produced by these tumors was (1 O@Fl and Fl 0) were cultured in 1251-fibrin-coated tubes for due to the production of PA (Experiment 10) and that a quan various periods of time. At the end of each culture interval, titative difference in PA production between Fl and Fl 0 does tubes were immediately centrifuged, and 0.2 ml of supematant exist. This PA-dependent FA of Fl and Fl 0 was further sup was counted to measure degraded 1251-fibrin.Within the first ported by another experiment (Table 4). As compared to con 12 hr of incubation, no difference was observed between Fl trol assay tubes containing no tumor cells, neither Fl nor Fl 0 and Fl 0 with respect to their PA production (Chart 2). However, showed FA when they were cultured in tubes containing no Fl 0 expressed a significantly higher level of FA than did Fl plasminogen. However, significant fibrinolysis was demon after they were incubated for 24 hr or more. Cell-free super strated by both tumor cells when 0.8 ng of plasminogen was natants harvested from Fl and Fl 0 which had been cultured added to tubes. Fl 0 again had a significantly higher FA than for various periods of time were similarly tested, and results did Fl (p < 0.001). were recorded in Chart 3. Again, culture supematants of Fl 0 Tumor Cell Number and PA Production. Various numbers cells that had been cultured for 24 hr or more had FA signifi of Fl and Fl 0 cells in 1 ml of complete medium were incubated cantly greater than those from Fl cells that had been incubated in 1251-fibrin-coatedtubesfor 24 hr, and the amount of degraded for the same period of time. These experiments also indicated â€˜251-fibrinwasthen determined. As indicated in Chart 1, 1O@to that 24 hr were necessary for both Fl and Fl 0 cells to produce 10@Fl 0 cells produced significantly higher FA as compared to significant amounts of PA. Furthermore, both tumors appeared the same number of Fl cells being tested. However, there was to have a similar doubling time since, over a period of 72 hr, no significant difference in FA between Fl and Fl 0 when 10@ the total numbers of cells did not significantly differ from each cells or fewer were tested. other as judged by counting the cells and measuring incorpo rated [3H@thymidmneinDNA of these cells (results not shown). Therefore, the difference in FA between Fl and Fl 0 was in Table 4 fact due to the different amounts of PA produced by these PA-dependent fibrinolysis by Fl and Fl 0 melanomas cells. of released â€˜[email protected] groupcpm cells (control) Â±79.1 DISCUSSION 30.0bN?C.FlOB.NoFl cellsC466.3 509.6 Â± 45.2NSD.celIsC474.0 Â± In this study, we have demonstrated that Bl 6 mets cells cells + plasminogen 106.0 Â±75.2 harvested from the pulmonary metastatic nodules of C57BL/ E.FlFl0cells+plasmmnogene11706.3 Â±56.2<0.001 <0.001 6J mice bearing the Bl 6 melanoma showed a higher FA in vitro a,@valuesweredeterminedbycomparingFAofeachexperimentalgroupwith that of Control Group A, which had no cells. The p value between Groups B and than did the Bl 6 cells from the primary tumors in the same C was 0.3, and the p value between Groups D and E was <0.001. mice (Table 1). Although about 80% of Bl 6 mets cells were b Mean Â±S.D. of triplicate cultures. shown to contain melanin, we could not completely rule out the C We tested 1 O@ tumor cells. d NS, not significant. possibility that the increased FA seen with Bl 6 mets might be e Plasminogen (0.8 ng) was added. caused by the presence of normal cells in the lung, some of

l8,QOO S 0 V z a.

@ 10,000

Chart 1. Cell number and PA production. Various numbers (102to ! 10@)of Fl (â€¢)andFl 0 (0) cells were incubated in â€˜25I-flbrin-coated culture tubes for 24 hr. The amount of â€˜26l-fibrindegradedfrom these @jj 7,500 tubes was determined from triplicate @iItures.Bars,S.D. CO w 0 -J 0 V @5,O0O a. LI 0 @2,5O0 0

0 ,@! lO@ lÃ ' lO@ l0 l0@ NUMBER OF TUMOR CELLS

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(20) demonstrated that fibrinolysis induced by human plasmin ogen in mice increased the incidence of metastases, whereas antifibrinolysis induced with tranexamic acid decreased metas 10,000 tases. Hagmar (9) enhanced hepatic and pulmonary metasta ses in mice by treatment with arvin, an agent which depletes fibrinogen. Furthermore, normal embryo fibroblast cultures usually did not produce FA unless they were transformed by oncogenic viruses (21 , 23, 25). Several malignant human and 8,000 nonhuman tumor cell lines were also shown to be highly fibrin z olytic in cultures (11â€”13,19). Although all these observations have repeatedly suggested that FA of a tumor may closely LI relate to its metastatic potential, results from the present study provide for the first time direct evidence and quantitative mea I 6,000 0 surements to support this hypothesis. Although Nicolson et a!. w (18) previously reported no difference in FA between 1O@Fl Cl) cells and the same number of Fl 0 cells as we confirmed in the present study, a significant difference in PA production be tween these 2 tumors was clearly demonstrated in vitro when @ 4,000 1O@ormore cells were tested (Chart 1). Laug et a!. (13) observed that malignant human tumor cell lines had a greater ability to induce fibrinolysis in vitro and that

U they also more frequently caused tumors in immunosuppressed animals in vivo than did normal cell lines. Pollack et a!. (21) 2,000 also found a consistent relationship of PA production of SV4O- transformed rat cell lines with plating efficiency in semisolid medium which was tightly correlated with tumorigenicity in nude mice as suggested by Freedman and Shin (7). However, Wolf and Goldberg (26) and Jones et a!. (1 1) failed to observe 0 it in other tumor systems. In this study, we did not examine the 36@ 24 48 72 relationship between PA production and tumorigenicity but INCUBATION TIME (HOURS) rather established a positive correlation between PA production Chart 2. TIme course of PA production by tumor cells. Fl (â€¢)andFlO (0) cells (1O@)werecultured In â€˜251-flbrin-coatedtubesfor various periods of time (0 to 72 hr). The amount of â€˜25l-fibrindegradedfrom these tubes atthe end of each culture Interval was determined from triplicate cultures. Bars, S.D.

which are known to be highly fibrinolytic. Therefore, we have subsequently examined 2 well-established sublines which were 6000 also isolated from metastatic pulmonary Bl 6 melanoma nod z ules, and we foundthat Fl 0 tumorcells consistentlyproduced @ PA in significantlylarger amounts than did Fl tumor cells. 5000 Since both Fl and Fl 0 were maintainedin the tissue culture for a large number of generations in our laboratory, the pres ence of normal lung cells in these 2 tumor sublines seemed to @D4000 w be extremely unlikely. Fidler (6) reported that, after excision of Cl) @ Fl and Fl 0 prImary isografts resulting from previous s.c. 3000 injections with 5 x 10@viable tumor cells, mice developed w-I pulmonary metastases in 6 to 8 weeks. The incidence of metastasisfor Fl was 9.1 %, as contrastedwith 75% for Fl 0. LA@2000 The present results (Table 2) not only confirm his observation 0 but also indicate a possible correlation between the FA of these 0@ 1000 tumors and their potential for metastasis. Furthermore, our C.) results suggest that a quantitative difference in PA production is responsible for the difference in FA observed (Tables 3 and 0 4). 2 24 48 72 Retik et al. (22) have reported that prolonged treatment with INCUBATION TIME (HOURS) hepann markedlyincreasedthe numberof metastasesin mice Chart 3. Time course of FA by cell-free supernatants harvested from tumor which were previously inoculated with sarcoma cells. A similar cultures. Ten x I 0' Fl (â€¢)andFl 0 (0) cells were cultured in 20 ml of medium increase in metastases by heparin treatment was also observed for various periods of time (0 to 72 hr). The supernatants were harvested. One ml of these supernatants was added to â€˜25i-flbrln-coated tubes and incubated for 24 by Boeryd (1) in mice which were transplanted with a chemi hr. The amount of â€˜251-flbrlndegradedfrom tubes was determined from triplicate cally-induced rhabdomyosarcoma. Peterson and Rudenstam cultures. Bars, S.D.

FEBRUARY1980 291

of a tumor and its metastatic potential. 5. Donati, M. B., Mussoni, L, Poggi, A., DeGaetano, G., and Garattmni,S. The successful establishment of a blood-borne metastasis Growth and metastasisof the Lewis lung carcinoma in mice defibrinated with batroxobin. Eur. J. Cancer, 14: 343â€”347,1978. seems to be dependent upon 2 steps: the release of tumor 6. Fidler, I. J. Biological behavior of malignant melanoma cells correlated to cells from the primary site and attachment of circulating tumor their survival in vivo. Cancer Res., 35: 218â€”224,1975. cells in the capillary bed of the secondary site. Although both 7. Freedman, V. H., and Shin, S-I. Cellular tumongenicity in nude mice: con's- lation with cell growth in semi-solid medium. Cell, 3: 355â€”359,1974. steps are essential, the latter cannot take place unless a 8. Grossl. C. E., Agostino, D., and Clinton, E. E. The effect of humanfibrinolysin sufficient number of tumor cells are released in the first place. on pulmonary metastases of Walker 256 carcinoma. Cancer Res., 20: 605â€” 608, 1960. Therefore, we believe that Fl 0 cells with a higher yield of PA 9. Hagmar, B. Defibrination and metastasis formation: Effects of arvin on are able to lyse surrounding fibrin deposits and thus can more experimental metastases in mice. Eur. J. Cancer, 8: 17â€”28,1972. easily spread from the primary site than can Fl cells which 10. Hammond, W. G., Fisher, J. C., and Rolley, R. T. Tumor-specific transplan tation immunity to spontaneous mouse tumors. Surgery (St. Louis), 62: 124â€” produce less PA. At the stage of tumor cell reattachment, on 133, 1967. the other hand, fibrinolysis has been shown to have the oppo 11. Jones, P. A., Lang. W. E., and Benedict, W. F. Fibrmnolyticactivity In a site effect (5). This was supported by studies of Cliffton and human fibrosarcoma cell line and evidence for the induction of plasminogen activator secretion during tumor formation. Cell, 6: 245â€”252,1975. Agostino (3), Grossi et a!. (8), and Brown (2), who injected 12. Jones, P. A., Rhim, J. S.. lsaacs, J., Jr., and McAllister, A. M. The relation tumor cells i.v. into animals and found that the number of ship between tumorigenicity, growth in agar and fibrmnolyticactivity In a line metastases was dramatically decreased when these animals of human osteosarcoma cells. Int. J. Cancer, 16: 616â€”621â€¢1975. 13. Lang, W. E., Jones, P. A., and Benedict, W. F. Relationship between were treated with heparin, fibrinolysin, or warfarin, suggesting fibrinolysis of cultured cells and malignancy. J. Nati. Cancer Inst., 54: 173â€” that FA may have prevented tumor cells in the bloodstream 179, 1975. 14. Lassen, M. Heat denaturation of plasminogen in the fibrin plate method. from adhering to capillary walls. Thus, it seems likely that, in Acta Physlol. Scand., 27: 371â€”376,1953. order to anchor successfully to the capillary walls in the lung, 15. Loskutoft, D. J.. and Edgington, T. S. Synthesis of a fibrinolytic activator Bl 6 melanoma cells must stop producing PA or they must and inhibitor by endothelial cells. Proc. Nati. Acad. Sci. U. S. A. 74: 3903â€” 3907, 1977. produce an inhibitor to neutralize PA activity. An inhibitor of PA 16. Moroz, L. A., and Gilmore, N. J. A rapid and sensitive â€˜25l-flbrinsolid-phase has been demonstrated in an SV4O-transformed human cell fibrinolytic assay for plasmin. Blood, 46: 543â€”553,1975. line (23), in rabbit endothelial cells (15), and in the sera from 17. Newstead, G. L., Griffiths, J. D., and Salsbury, A. J. Fibrinolytic activity of carcinoma of the colorectum. Surg. Gynecol. Obst., 143: 61â€”64,1976. tumor-bearing chickens but not from normal chickens (25). 18. Nicolson, G., Winkelhake, J. L., and Nussey, A. C. An approach to studying Whether or not an inhibitor of PA plays a role in the metastatic the cellular properties associated with metastasis: some in vitro properties of tumor variants selected in vivo for enhanced metastasis. In: L Weiss (ed.), melanoma system studied in the present experiments is not Fundamental Aspects of Metastasis, pp. 291-303. Amsterdam: North-Hol clear, and this investigation is currently under way in our land Publishing Co., 1976. laboratory. 19. Peterson, H-I., Kjartansson, I., Korsan-Bengtsen, K., Rudenstam, C-M., and Zettergren, L. Fibrinolysis in human malignant tumors. Acta Chir. Scand., 139: 219â€”223, 1973. 20. Peterson, H-I., and Rudenstam, C-M. Influence of induced fibrinolysis and ACKNOWLEDGMENTS antifibrinolysis on tumour growth and on spontaneous metastasis formation of the mammary carcinoma. Acta Chir. Scand. (Suppl.), 394: 21â€”26,1968. We wish to thank S. Onikul, I. Saporoschetz, K. Collins, and E. Heacock for 21. Pollack, R., Risser, A., Conlon, S., and Rifkin. D. Plasminogen activator their technical assistance and S. Quimby and D. DePinafor their secretarial help. production accompanies loss of anchorage regulation In transformation of Valuable advice and criticism from Dr. Andrew Wu are also appreciated. primary rat embryo cells by Simian Virus 40. Proc. Nati. Aced. Sd. U. S. A., 71: 4792â€”4796,1974. 22. Retik,A.B..Arons,M.S.,Ketcham,A.S.,andMantel,N.Theeffectof heparin on primary tumors and metastases. J. Surg. Res., 2: 49â€”53,1962. REFERENCES 23. RoblIn, R. 0., Young, P. L., and Bell, T. E. Concomitant secretion by transformed SVWI38-VA13-2RA cells of plasminogen activator(s) and sub 1. Boeryd, B. Action of heparin and plasminogen inhibitor (EACA)on metastatic stance(s) which prevent their detection. Blochem. Blophys. Res. Commun., tumour spread in an isologous system. Acta Pathol. Micribiol. Scand., 65: 82: 165â€”172,1978. 395-404, 1965. 24. Tucker, W. S., Kirsch, W. M., Martinex-Hernandez, A., and Fink, L. M. In 2. Brown, J. M. A study of the mechanism by which anticoagulation with vitro plasminogen activator activity in human brain tumors. Cancer Rca., 38: warfarin inhibits blood-borne metastases. Cancer Res., 33: 1217â€”1224, 297â€”302, 1978. 1973. 25. Unkeless, J. C., Tobia, A., Ossowski, L, Quigley, J. P., Rlfkin, D. B., and 3. Cliffton, E. E., and Agostino, D. The effects of fibrin formation and alterations Reich, E. An enzymatic function associated with transformation of fibroblasts in the clotting mechanism on the development of metastases. Vasc. Dis.. 2: by oncogenic virus. I. Chick embryo fibroblast cultures transformed by avian 43â€”52,1965. RNAtumorviruses.J.Exp.Med.,137:85-111â€¢1973. 4. Deutsch, D. G., and Mertz, E. T. Plasminogen: purification from human 26. Wolf, B. A., and Goldberg, A. R. Lack of correlation between tumongenicity plasma by affinity chromatography. Science (wash. D. C.), 170: 1095â€” and level of plasminogen activator in fibroblasts transformed by Rous sar 1096, 1970. coma virus. Proc. NatI. Acad. Sd. U. S. A., 75: 4967â€”4971,1978.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1980 American Association for Cancer Research. Correlation of the Production of Plasminogen Activator with Tumor Metastasis in B16 Mouse Melanoma Cell Lines

Bosco Shang Wang, Gerard A. McLoughlin, Jerome P. Richie, et al.

Cancer Res 1980;40:288-292.

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